PROJECT SUMMARY/ABSTRACT
Proper localization of RNA is critical for proper RNA function, downstream protein localization, cell function,
organism development, and organism health. Of particular interest, proper sequestration of RNA into
ribonucleoprotein (RNP) granules helps cells respond efficiently to stress, while persistent RNA localization to
RNP granules may serve as nucleation points for proteins implicated in ALS, Alzheimer’s, and dementia. During
stress, stress granules (SGs) sequester RNA from translational machinery while processing bodies (PBs)
degrade RNA. SGs and PBs dock, or interact, in certain stress conditions and one stressor has been shown to
promote transfer of mRNA between the two RNP granules. SG-PB docking and mRNA transfer is hypothesized
to function as an additional regulatory measure during stress. mRNA transfer is hypothesized to be facilitated by
specific interactions with two RNA-binding proteins, TTP and CPEB1. However, both hypotheses remain largely
untested. In order to identify RNA localization in healthy or stressed and disease states, scientists use RNA-
imaging tools. Tools that allow us to see single molecules of RNA in live cells are of particular value since biology
is incredibly heterogenous and responds to stimuli in real time. The tool historically used to monitor mRNA
dynamics in RNP granules uses many fluorescent proteins to visualize RNA. Since RNP granules form largely
by nonspecific interactions between proteins and RNAs, this tool may compromise some of our conclusions
about the resultant data. A tool that uses small molecule fluors to visualize RNA may be better suited to
investigate this question. Riboglow was developed as a collaboration between my lab, a fluorescent tool lab, and
an RNA-biology lab. Riboglow is a live-cell RNA-imaging tool that is comprised of two main parts: an RNA
aptamer that is appended to an RNA of interest and a small-molecule fluorescent probe. Riboglow outperformed
the gold-standard tool at detecting mRNA recruitment to stress granules and preliminary data shows that
Riboglow can achieve single-molecule detection of mRNA in live cells. However, Riboglow remains a new tool.
In this proposal, I will 1) optimize and characterize Riboglow for single-molecule detection in live cells, 2) quantify
any effects that tagging an RNA with Riboglow has on basic RNA biology, 3) use Riboglow to establish a list of
stressors that cause SG-PB docking and mRNA transfer, and 4) evaluate the hypothesis that mRNA transfer is
facilitated by specific interactions with TTP and CPEB1. My research will benefit science by expanding the single-
molecule live-cell RNA-imaging toolbox for RNA biologists and by yielding foundational knowledge for the RNP
field. This will facilitate further studies on RNA localization in response to stimuli and the potential relationship
between neurodegenerative diseases and environmental stressors. This fellowship will provide me with
advanced training in biochemical tool development and optimization, quantitative fluorescence microscopy for
biological studies, and data science for image analysis. This interdisciplinary set of skills will position me to
achieve my career goals and positively impact the biomedical field.